The pathways of folic acid metabolism are complex and incompletely understood in eucaryotes, but the integrity of at least major portions of these pathways is clearly necessary for the viability and normal development. Over the past 3 years we have demonstrated the presence in experimentally useful amounts of 9 of the 15 major enzymes of folate metabolism in a variety of cultured animal cells, and have made substantial progress toward our ultimate goal of understanding the biochemical-genetic regulation of both individual folate enzymes and the pathways as a whole. This system has allowed us to measure accurately the folate activities under a variety of culture conditions in both normal or wild-type cells and in cells thought to have specific mutations, including those from patients with putative inborn errors of folate metabolism and mutant sublines produced and/or selected in vitro. Using recently developed techniques we have also begun to supplement the information gained from the measurement of enzyme activities with data on the quantitative distribution of intracellular folate intermediates under a variety of culture conditions in normal and mutant cells. We propose here specific experiments to pursue particularly promising leads in order to characterize in considerably greater detail than possible previously the biochemical-genetic regulation of the folate pathways in normal and mutants, latter including patients having inborn errors of folate metabolism leading to clinical abnormalities. We also propose to continue our studies of lysylprotocollagen hydroxylase deficiency, the first true inborn error of human collagen metabolism in which the affected enzyme and product are known, to gain further understanding of the nature of the basic defect and the resulting clinical abnormalities, and in a search for other collagen inborn errors.